Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity

BACKGROUND: Host plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings. RESULTS: Specific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots. CONCLUSIONS: Real-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth

Metropolis versus necropolis: Polarity in the relationship between the city and the cemetery in history

The evolution of the city and the cemetery have always been running parallel in history, one couldn't exist without the other their relationship has always been strongly intertwined, yet strongly polarised. The aim of this report is to analyse key historical examples, relating to particular historical events, which in my opinion, may help to open - at least a partial view - on the evolution of the relationship between the city and the cemetery, affected by the shifting and changing balance of religious, political and socio-cultural components. In ancient times, the human body was taken as a reference to design the city first developing the metaphor of the city as a living organism - a metaphor which will be largely used through history, as will we see later. In the Greek and Roman cultures, the dead were not allowed to pollute the space of the living and the cemetery and the city were two separate entities. The advent of Christianity in Medieval times, saw the introduction of churchyards within the city walls, which also along with poor heath conditions of the then cities, facilitated the spread of epidemic diseases such as cholera. The Renaissance brought with it the desire to start afresh new cities and towns. The many projects of ideal cities left behind by architects such as Filarete or Scamozzi, were primarily concerned with the aesthetic and the security of the city rather than the real issues- health and hygiene and the spread epidemic diseases issues that finally urbanists had to address with the expansion of the cities due to the advent of the Industrial Revolution. Work forces during this particular historical moment moved from the countryside to city centres. The absence of any burial regulation and health issues urged the introduction of legislations which moved the cemeteries out of the city. In England, the Burial Act (which came to force in 1851) marked the introduction of suburban necropolises in the Victorian era, to resolve the issues of crowed inner city burial grounds, and guarantee city inhabitants a healthier life in the city centres. This also facilitated the condition for the development of a new strong architectural language specific to the cemetery in the Victorian era, which mostly came from cultures with strong past records of funerary traditions, like the Egyptians or the Romans. The new suburban necropolis in Victorian England, was the starting point of a new type of cemetery, which was also perceived as a public civic space, like a park. From now on we will see that the city and the cemetery will be almost completely separated: the city - growing into the metropolis, a complex, sometimes chaotic large urban development, and the cemetery - The Renaissance brought with it the desire to start afresh new cities and towns. The many projects of ideal cities left behind by architects such as Filarete or Scamozzi, were primarily concerned with the aesthetic and the security of the city rather than the real issues health and hygiene and the spread epidemic diseases issues that finally urbanists had to address with the expansion of the cities due to the advent of the Industrial Revolution. Work forces during this particular historical moment moved from the countryside to city centres. The absence of any burial regulation and health issues urged the introduction of legislations which moved the cemeteries out of the city. In England, the Burial Act (which came to force in 1851) marked the introduction of suburban necropolises in the Victorian era, to resolve the issues of crowed inner city burial grounds, and guarantee city inhabitants a healthier life in the city centres. This also facilitated the condition for the development of a new strong architectural language specific to the cemetery in the Victorian era, which mostly came from cultures with strong past records of funerary traditions, like the Egyptians or the Romans. The new suburban necropolis in Victorian England, was the starting point of a new type of cemetery, which was also perceived as a public civic space, like a park. From now on we will see that the city and the cemetery will be almost completely separated: the city - growing into the metropolis, a complex, sometimes chaotic large urban development, and the cemetery

Impact of mycorrhiza helper bacterium Streptomyces sp. AcH 505 on the genetic and physiuological regulation in oaks associated to pathogenic and symbiotic fungi

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant

Impact of mycorrhiza helper bacterium Streptomyces sp. AcH 505 on the genetic and physiuological regulation in oaks associated to pathogenic and symbiotic fungi

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant

Streptomyces-Elicited Defence Response of Oak (Quercus robur) To Powdery Mildew Infection

Streptomyces can be favourable to plant growth in several ways. In their presence, the formation and functioning of mycorrhiza, a mutually beneficial plant-fungus symbiosis, may be promoted. In contrast, production of secondary metabolites can repress fungal growth at plant roots, thus preventing root colonisation. Most interestingly however, root colonisation with streptomycetes can trigger a response that is detectable in distal plant parts, i.e. in the leaves. Here we show that inoculation of roots of pedunculate oak (Quercus robur) with the Mycorrhiza Helper Bacterium Streptomyces sp. AcH 505 can be observed in oak leaves as a specific modulation of gene expression. Concomitantly, the plants’ defence response against the leaf pathogenic fungus Microsphaera alphitoides (oak powdery mildew) is increased, alleviating the detrimental effect of the fungal infection. Quercus robur clone DF159, the clonal tree model of the platform TrophinOak [1], was used to investigate how oak coordinates these biotrophic interactions.The analysis of gene expression patterns based on RNA-seq in the oak- Streptomyces AcH 505-powdery mildew interaction suggests that Streptomyces AcH 505 not only activates the jasmonic acid / ethylene dependent defence response, as it has previously been described in plant-bacterium interactions that result in increased resistance. Instead, Streptomyces AcH 505 also modulates salicylic acid and abscisic acid dependent signalling, even in the absence of the fungal pathogen. These signalling pathways may be activated as part of a direct defence against pathogen attack, suggesting that Streptomyces AcH 505 is perceived by the plant as a potential pathogen. This view was further substantiated following the comparison of gene expression patterns in single inoculations with powdery mildew and Streptomyces AcH 505, respectively. Here we were surprised to recognise that the systemic responses of oak to the fungus and to Streptomyces AcH 505 share patterns of gene expression [2]. This study provides novel insights into the molecular mechanisms of priming in plants for enhanced defence and may be instrumental in understanding actinobacteria elicited defence responses. In the case of Streptomyces AcH 505, priming acts in concert with proven antibiotics production and exudation of stimulators of mycorrhiza formation, and underlines the versatility of Streptomyces bacteria upon interactions with eukaryotes.References[1] Herrmann et al. Perspectives in Plant Ecology, Evolution and Systematics. 2016, 19:40-48[2] Kurth et al. Molecular Plant-Microbe Interactions. 2014, 27:891-90

Ectomycorrhizal fungus supports endogenous rhythmic growth and corresponding resource allocation in oak during various below- and aboveground biotic interactions

Endogenous rhythmic growth (ERG) is displayed by many tropical and some major temperate tree species and characterized by alternating root and shoot flushes (RF and SF). These flushes occur parallel to changes in biomass partitioning and in allocation of recently assimilated carbon and nitrogen. To address how biotic interactions interplay with ERG, we cross-compared the RF/SF shifts in oak microcuttings in the presence of pathogens, consumers and a mycorrhiza helper bacterium, without and with an ectomycorrhizal fungus (EMF), and present a synthesis of the observations. The typical increase in carbon allocation to sink leaves during SF did not occur in the presence of root or leaf pathogens, and the increase in nitrogen allocation to lateral roots during RF did not occur with the pathogens. The RF/SF shifts in resource allocation were mostly restored upon additional interaction with the EMF. Its presence led to increased resource allocation to principal roots during RF, also when the oaks were inoculated additionally with other interactors. The interactors affected the alternating, rhythmic growth and resource allocation shifts between shoots and roots. The restoring role of the EMF on RF/SF changes in parallel to the corresponding enhanced carbon and nitrogen allocation to sink tissues suggests that the EMF is supporting plants in maintaining the ERG